Alzheimer’s disease (AD) is characterized by two hallmark pathologies: amyloid beta plaques and tau tangles. However, up to 70% of AD cases also have abnormal clumps of a protein called TDP-43, a pathology usually associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). These cases, classified as AD with ‘Limbic-predominant Age-related TDP-43 Encephalopathy Neuropathologic Changes’ (LATE-NC), tend to have worse clinical outcomes than AD alone. This suggests that TDP-43 contributes to AD—yet how remains unclear.
TDP-43 is found in the cell’s nucleus, where it regulates gene expression through RNA splicing. Splicing involves cutting and joining different parts of the genetic code together to create a final RNA transcript that serves as the instructions for building a protein. It is like editing a film—some scenes are cut, and others are joined together to create the final film. TDP-43 acts as the quality control in this process. One of its roles is to prevent hidden (aka cryptic) sections of RNA from being included in the final transcript. If it fails, cryptic mis-splicing occurs. Most often, if a cryptic transcript is made, it is immediately degraded. However, sometimes a cryptic transcript slips through, and an abnormal protein is made, which can lead to neurodegeneration. Understanding how faulty RNA splicing impacts Alzheimer’s, and the role that TDP-43 plays, may help in the development of new therapies that reverse cryptic mis-splicing and slow or prevent disease.
Dr. Petrucelli’s team and others found high levels of three specific cryptic transcripts (STMN2, UNC13A, and HDGFL2) in brain regions with TDP-43 pathology in AD/LATE-NC patients. STMN2 and UNC13A are important for neuron health and function, while HDGFL2 is involved in DNA repair. Cryptic mis-splicing transcripts of STMN2 and UNC13A are degraded, which results in fewer of these proteins being made. However, cryptic transcripts of HDGFL2 avoid degradation and are used to make faulty HDGFL2 proteins. To detect the faulty HDGFL2 protein, Dr. Petrucelli has developed a novel antibody tool. His team also has a strong record of creating new laboratory tools not only for their applications but for other scientists to use as well. For example, they engineered a mouse model with a mutated form of TDP-43 that impairs RNA binding and splicing, which is useful for testing approaches to reverse cryptic mis-splicing caused by TDP-43. Dr. Petrucelli hypothesizes that TDP-43 pathology causes cryptic mis-splicing of STMN2, UNC13A, and HDGFL2 and that these changes affect disease severity in AD/LATE-NC. He specifically predicts that loss of STMN2 and UNC13A proteins causes neuron dysfunction and degeneration, while an increase in cryptic HDGFL2 protein impairs the cell’s ability to repair DNA damage.
Dr. Petrucelli’s team proposed three aims. In the first aim, they are determining if the proteins encoded by the STMN2, UNC13A and HDGFL2 genes are impacted by RNA mis-splicing in AD/LATE-NC. This is an important step in determining whether cryptic mis-splicing is having a functional impact; after all, it is the protein, not the RNA transcript, that is actually doing the work in the cell. They are measuring protein levels in post-mortem samples from AD patient brains with and without TDP-43 pathology, as well as in age-matched, cognitively intact controls. In the second aim, they are testing their hypothesis that cryptic mis-splicing of STMN2, UNC13A and HDGFL2 triggers synaptic dysfunction, neurodegeneration and increased DNA damage, respectively. First, they are experimentally lowering STMN2 and UNC13A in control mice and their TDP-43 mutant mouse model. They predict that lowering both will negatively impact behavior, increase neuroinflammation and result in neurodegeneration and synapse loss, and that these effects are worse in the mutant TDP-43 mice. Then, they are determining if HDGFL2 is associated with DNA damage repair processes in human tissue. They are also improving their antibody tools to better detect the cryptic HDGFL2 protein, and co-labeling AD/LATE-NC brain sections for markers of DNA damage. In the third aim, they are modeling and trying to reverse cryptic mis-splicing defects in mice. This requires a novel mouse model created by breeding their TDP-43 mutant line with a mouse engineered to have the human STMN2 gene (because the mouse version is not a TDP-43 target.) Behavioral testing and markers of neurodegeneration will be measured in these mice at several ages. Finally, assuming these mice exhibit pathological outcomes, the Petrucelli team is testing an innovative gene therapy approach (U7-snRNA), which has only been tested in cell cultures to date, that can alter the RNA splicing of specific genes.
In the first year, the team has made strong progress across all three aims. In aim one, the team found that HDGFL2-CE, a protein that only appears when TDP-43 malfunctions, is elevated and closely tracked with disease pathology, suggesting it could serve as a new biomarker. They are developing sensitive new tools to detect this protein and, moving forward, will continue measuring HDGFL2-CE and related cryptic proteins in human brain tissue to strengthen its potential as a disease marker. In the second aim, they are testing how loss of the proteins STMN2 and UNC13A impacts brain health in mice. Early experiments indicate that lowering these proteins affects behavior and brain health, but the methods need refining, and the team is now improving their models and approaches to generate more robust results. In aim three, they successfully created a new mouse model to test whether repairing RNA errors with the novel gene therapy (U7-snRNA) can reverse disease-related changes. Over the next year, they will run long-term behavioral and pathology studies in this model to assess whether the therapy can rescue cognitive and cellular deficits.
